Transgenic microbial polyhydroxyalkanoate producers

ABSTRACT

Transgenic microbial strains are provided which contain the genes required for PHA formation integrated on the chromosome. The strains are advantageous in PHA production processes, because (1) no plasmids need to be maintained, generally obviating the required use of antibiotics or other stabilizing pressures, and (2) no plasmid loss occurs, thereby stabilizing the number of gene copies per cell throughout the fermentation process, resulting in homogeneous PHA product formation throughout the production process. Genes are integrated using standard techniques, preferably transposon mutagenesis. In a preferred embodiment wherein mutiple genes are incorporated, these are incorporated as an operon. Sequences are used to stabilize mRNA, to induce expression as a function of culture conditions (such as phosphate concentration), temperature, and stress, and to aid in selection, through the incorporation of selection markers such as markers conferring antibiotic resistance.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Priority is claimed to U.S. provisional application Serial No.60/096,852, filed Aug. 18, 1998, the teachings of which are incorporatedherein.

BACKGROUND OF THE INVENTION

[0002] The present invention is generally in the field of biosynthesisof poly(3-hydroxyalkanoates), and more particularly to improvedmicrobial strains useful in commercial production ofpolyhydroxyalkanoates.

[0003] Poly(3-hydroxyalkanoates) (PHAs) are biological polyesterssynthesized by a broad range of bacteria. These polymers arebiodegradable and biocompatible thermoplastic materials, produced fromrenewable resources, with a broad range of industrial and biomedicalapplications (Williams & Peoples, CHEMTECH 26:38-44 (1996)). PHAbiopolymers have emerged from what was originally considered to be asingle homopolymer, poly-3-hydroxybutyrate (PHB) into a broad class ofpolyesters with different monomer compositions and a wide range ofphysical properties. About 100 different monomers have been incorporatedinto the PHA polymers (Steinbuchel & Valentin, FEMS Microbiol. Lett.128:219-28 (1995)).

[0004] It has been useful to divide the PHAs into two groups accordingto the length of their side chains and their biosynthetic pathways.Those with short side chains, such as PHB, a homopolymer ofR-3-hydroxybutyric acid units, are crystalline thermoplastics, whereasPHAs with long side chains are more elastomeric. The former have beenknown for about seventy years (Lemoigne & Roukhelman, 1925), whereas thelatter materials were discovered relatively recently (deSmet et al., J.Bacteriol. 154:870-78 (1983)). Before this designation, however, PHAs ofmicrobial origin containing both (R)-3-hydroxybutyric acid units andlonger side chain (R)-3-hydroxyacid units from C₅ to C₁₆ had beenidentified (Wallen & Rohweder, Environ. Sci. Technol. 8:576-79 (1974)).A number of bacteria which produce copolymers of (R)-3-hydroxybutyricacid and one or more long side chain hydroxyacid units containing fromfive to sixteen carbon atoms have been identified (Steinbuchel & Wiese,Appl. Microbiol. Biotechnol. 37:691-97 (1992); Valentin et al., Appl.Microbiol. Biotechnol. 36:507-14 (1992); Valentin et al., Appl.Microbiol. Biotechnol. 40:710-16 (1994); Abe et al., Int. J. Biol.Macromol. 16:115-19 (1994); Lee et al., Appl. Microbiol. Biotechnol.42:901-09 (1995); Kato et al., Appl. Microbiol. Biotechnol. 45:363-70(1996); Valentin et al., Appl. Microbiol. Biotechnol. 46:261-67 (1996);U.S. Pat. No. 4,876,331 to Doi). A combination of the two biosyntheticpathways outlined described above provide the hydroxyacid monomers.These copolymers can be referred to as PHB-co-HX (where X is a3-hydroxyalkanoate or alkanoate or alkenoate of 6 or more carbons). Auseful example of specific two-component copolymers isPHB-co-3-hydroxyhexanoate (PHB-co-3HH) (Brandl et al., Int. J. Biol.Macromol. 11:49-55 (1989); Amos & Mclnerey, Arch. Microbiol. 155:103-06(1991); U.S. Pat. No. 5,292,860 to Shiotani et al.).

[0005] PHA production by many of the microorganisms in these referencesis not commercially useful because of the complexity of the growthmedium, the lengthy fermentation processes, or the difficulty ofdown-stream processing of the particular bacterial strain. Geneticallyengineered PHA production systems with fast growing organisms such asEscherichia coli have been developed. Genetic engineering also allowsfor the improvement of wild type PHA production microbes to improve theproduction of specific copolymers or to introduce the capability toproduce different PHA polymers by adding PHA biosynthetic enzymes havingdifferent substrate-specificity or even kinetic properties to thenatural system. Examples of these types of systems are described inSteinbuchel & Valentin, FEMS Microbiol. Lett. 128:219-28 (1995). PCT WO98/04713 describes methods for controlling the molecular weight usinggenetic engineering to control the level of the PHA synthase enzyme.Commercially useful strains, including Alcaligenes eutrophus (renamed asRalstonia eutropha), Alcaligenes latus, Azotobacter vinlandii, andPseudomonads, for producing PHAs are disclosed in Lee, Biotechnology &Bioengineering 49:1-14 (1996) and Braunegg et al., (1998), J.Biotechnology 65: 127-161.

[0006] The development of recombinant PHA production strains hasfollowed two parallel paths. In one case, the strains have beendeveloped to produce copolymers, a number of which have been produced inrecombinant E. coli. These copolymers includepoly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV),poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB),poly(4-hydroxybutyrate) (P4HB) and long side chain PHAs comprising3-hydroxyoctanoate units (Madison and Huisman, 1999. Strains of E. colicontaining the phb genes on a plasmid have been developed to produceP(3HB-3HV) (Slater, et al., Appl. Environ. Microbiol. 58:1089-94 (1992);Fidler & Dennis, FEMS Microbiol. Rev. 103:231-36 (1992); Rhie & Dennis,Appl. Environ. Micobiol. 61:2487-92 (1995); Zhang, H. et al., Appl.Environ. Microbiol. 60:1198-205 (1994)). The production of P(4HB) andP(3HB-4HB) in E. coli is achieved by introducing genes from ametabolically unrelated pathway into a P(3HB) producer (Hein, et al.,FEMS Microbiol. Lett. 153:411-18 (1997); Valentin & Dennis, J.Biotechnol. 58:33-38 (1997)). E. coli also has been engineered toproduce medium short chain polyhydroxyalkanoates (msc-PHAs) byintroducing the phaC1 and phaC2 gene of P. aeruginosa in a fadB::kanmutant (Langenbach, et al., FEMS Microbiol. Lett. 150:303-09 (1997); Qi,et al., FEMS Microbiol. Lett. 157:155-62 (1997)).

[0007] Although studies demonstrated that expression of the A. eutrophusPHB biosynthetic genes encoding PHB polymerase, -ketothiolase, andacetoacetyl-CoA reductase in E. coli resulted in the production of PHB(Slater, et al., J. Bacteriol. 170:4431-36 (1988); Peoples & Sinskey, J.Biol. Chem. 264:15298-303 (1989); Schubert, et al., J. Bacteriol.170:5837-47 (1988)), these results were obtained using basic cloningplasmid vectors and the systems are unsuitable for commercial productionsince these strains lacked the ability to accumulate levels equivalentto the natural producers in industrial media.

[0008] For commercial production, these strains have to be made suitablefor large scale fermentation in low cost industrial medium. The firstreport of recombinant P(3HB) production experiments in fed-batchcultures used an expensive complex medium, producing P(3HB) to 90 g/L in42 hours using a ph-stat controlled system (Kim, et al., Biotechnol.Lett. 14:811-16 (1992)). Using stabilized plasmids derived from eithermedium- or high-copy-number plasmids, it was shown that E. coli XL1-Bluewith the latter type plasmid is required for substantial P(3HB)accumulation (Lee, et al., Ann. N.Y. Acad. Sci. 721:43-53 (1994)). In afed-batch fermentation on 2% glucose/LB medium, this strain produced 81%P(3HB) at a productivity of 2.1 g/L-hr (Lee, et al., J. Biotechnol.32:203-11 (1994)). The P(3HB) productivity was reduced to 0.46 g/L-hr inminimal medium, but could be recovered by the addition of complexnitrogen sources such as yeast extract, tryptone, casamino acids, andcollagen hydrolysate (Lee & Chang, Adv. Biochem. Eng. Biotechnol.52:27-58 (1995); Lee, et al., J. Ferment. Bioeng. 79:177-80 (1995)).

[0009] Although recombinant E. coli XL1-blue is able to synthesizesubstantial levels of P(3HB), growth is impaired by dramaticfilamentation of the cells, especially in defined medium (Lee, et al.,Biotechnol. Bioeng. 44:1337-47 (1994); Lee, Biotechnol. Lett. 16:1247-52(1994); Wang & Lee, Appl. Environ. Microbiol. 63:4765-69 (1997)). Byoverexpression of FtsZ in this strain, biomass production was improvedby 20% and P(3HB) levels were doubled (Lee & Lee, J. Environ. PolymerDegrad. 4:131-34 (1996)). This recombinant strain produced 104 g/LP(3HB) in defined medium corresponding to 70% of the cell dry weight.The volumetric productivity of 2 g/L-hr, however, is lower thanachievable with R. eutropha. Furthermore, about 15% of the cells losttheir ability to produce PHB by the end of the fermentation (Wang & Lee,Biotechnol. Bioeng. 58:325-28 (1998)).

[0010] Recombinant E. coli P(3HB-3HV) producers reportedly are unable togrow to a high density and therefore are unsuited for commercialprocesses (Yim, et al., Biotechnol. Bioeng. 49:495-503 (1996)). In anattempt to improve P(3HB-3HV) production in a recombinant strain, fourE. coli strains (XL1-Blue, JM109, HB101, and DH5α) were tested by Yim etal. All four recombinant E. coli strains synthesized P(3HB-3HV) whengrown on glucose and propionate with HV fractions of 7% (Yim, et al.,Biotechnol. Bioeng. 49:495-503 (1996)). Unlike other strains studied(Slater, et al., Appl. Environ. Microbiol. 58:1089-94 (1992)),recombinant XL1-Blue incorporates less than 10% HV when the propionicacid concentration is varied between 0 and 80 mM. HV incorporation andPHA formation were increased by pre-growing cells on acetate followed byglucose/propionate addition at a cell density of around 10⁸ cells perml. Oleate supplementation also stimulated HV incorporation. Thisrecombinant XL1-Blue when pregrown on acetate and with oleatesupplementation reached a cell density of 8 g/L, 75% of which wasP(3HB-3HV) with an HV fraction of 0.16 (Yim, et al., Biotechnol. Bioeng.49:495-503 (1996)).

[0011] One of the challenges of producing P(3HB) in recombinantorganisms is the stable and constant expression of the phb genes duringfermentation. Often P(3HB) production by recombinant organisms ishampered by the loss of plasmid from the majority of the bacterialpopulation. Such stability problems may be attributed to the metabolicload exerted by the need to replicate the plasmid and synthesize P(3HB),which diverts acetyl-CoA to P(3HB) rather than to biomass. In addition,plasmid copy numbers often decrease upon continued fermentation becauseonly a few copies provide the required antibiotic resistance or preventcell death by maintaining parB. For these reasons, a runaway plasmid wasdesigned to suppress the copy number of the plasmid at 30 C and induceplasmid replication by shifting the temperature to 38 C (Kidwell, etal., Appl. Environ. Microbiol. 61:1391-98 (1995)). Using this system,P(3HB) was produced to about 43% of the cell dry weight within 15 hoursafter induction with a volumetric production of 1 gram P(3HB) per literper hour. Although this productivity is of the same order of magnitudeas natural P(3HB) producers, strains harboring these parB-stabilizedrunaway replicons still lost the capacity to accumulate P(3HB) duringprolonged fermentations.

[0012] While the instability of the phb genes in high cell-densityfermentations affects the PHA cost by decreasing the cellular P(3HB)yields, the cost of the feedstock also contributes to the comparativelyhigh price of PHAs. The most common substrate used for P(3HB) productionis glucose. Consequently, E. coli and Klebsiella strains have beenexamined for P(3HB) formation on molasses, which cost 33-50% less thanglucose (Zhang, et al., Appl. Environ. Microbiol. 60:1198-1205 (1994)).The main carbon source in molasses is sucrose. Recombinant E. coli andK. aerogenes strains carrying the phb locus on a plasmid grown inminimal medium with 6% sugarcane molasses accumulated P(3HB) toapproximately 3 g/L corresponding to 45% of the cell dry weight. Whenthe K. aerogenes was grown fed-batch in a 10 L fermenter on molasses asthe sole carbon source, P(3HB) was accumulated to 70% its cell dryweight, which corresponded to 24 g/L. Although the phb plasmid in K.aerogenes was unstable, this strain shows promise as a P(3HB) produceron molasses, especially since fadR mutants incorporate 3HV up to 55% inthe presence of propionate (Zhang, et al., Appl. Environ. Microbiol.60:1198-1205 (1994)).

[0013] U.S. Pat. No. 5,334,520 to Dennis discloses the production of PHBin E. coli transformed with a plasmid containing the phbCAB genes. Arec⁻, lac⁺ E. coli strain was grown on whey and reportedly accumulatesPHB to 85% of its cell dry weight. U.S. Pat. No. 5,371,002 to Dennis etal. discloses methods to produce PHA in recombinant E. coli using a highcopy number plasmid vector with phb genes in a host that expresses theacetate genes either by induction, constitutively, or from a plasmid.U.S. Pat. No. 5,512,456 to Dennis discloses a method for production andrecovery of PHB from transformed E. coli strains. These E. coli strainsare equipped with a vector containing the phb genes and a vectorcontaining a lysozyme gene. High copy number plasmids or runawayreplicons are used to improve productivity. The vectors are stabilizedby parB or by supF/dnaB(am). Using such strains, a productivity of 1.7g/L-hr was obtained corresponding to 46 g/L PHB in 25 hrs, after whichthe plasmid was increasingly lost by the microbial population. PCTWO94/21810 discloses the production of PHB in recombinant strains of E.coli and Klebsiella aerogenes with sucrose as a carbon source. PCT WO95/21257 discloses the improved production of PHB in transformedprokaryotic hosts. Improvements in the transcription regulatingsequences and ribosome binding site improve PHB formation by the plasmidbased phb genes. The plasmid is stabilized by the parB locus. PHBproduction by this construct is doubled by including the 361 nucleotidesthat are found upstream of phbC in R. eutropha instead of only 78nucleotides. It is generally believed that PHB production by recombinantmicroorganisms requires high levels of expression using stabilizedplasmids. Since plasmids are available in the cell in multiple copies,ranging from one to several hundreds, the use of plasmids ensured thepresence of multiple copies of the genes of interest. Since plasmids maybe lost, stabilization functions are introduced. Such systems, which aredescribed above, have been tested for PHB production, and the utility ofthese systems in industrial fermentation processes has beeninvestigated. However, overall PHB yield is still affected by loss ofphb genes.

[0014] It is therefore an object of the present invention to providerecombinant microorganisms strains useful in industrial fermentationprocesses which can accumulate commercially significant levels of PHBwhile providing stable and constant expression of the phb genes duringfermentation.

[0015] It is another object of the present invention to providetransgenic microbial strains for enhanced production ofpoly(3-hydroxyalkanoates).

[0016] It is another object of the present invention to providetransgenic microbial strains which yield stable and constant expressionof the phb genes during fermentation and accumulate commerciallysignificant levels of PHB, and methods of use thereof.

SUMMARY OF THE INVENTION

[0017] Transgenic microbial strains are provided which contain the genesrequired for PHA formation integrated on the chromosome. The strains areadvantageous in PHA production processes, because (1) no plasmids needto be maintained, generally obviating the required use of antibiotics orother stabilizing pressures, and (2) no plasmid loss occurs, therebystabilizing the number of gene copies per cell throughout thefermentation process, resulting in homogeneous PHA product formationthroughout the production process. Genes are integrated using standardtechniques, preferably transposon mutagenesis. In a preferred embodimentwherein mutiple genes are incorporated, these are incorporated as anoperon. Sequences are used to stabilize mRNA, to induce expression as afunction of culture conditions (such as phosphate concentration),temperature, and stress, and to aid in selection, through theincorporation of selection markers such as markers conferring antibioticresistance.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a diagram showing the construction of pMNXTp₁kan,pMNXTp₁cat, pMSXTp₁kan, and pMSXTp₁cat.

[0019]FIG. 2 is a diagram showing the construction of pMUXC₅cat.

[0020]FIG. 3 is a diagram showing the construction of pMUXAB₅cat,pMUXTp₁AB₅kan, pMUXTp₁₁AB₅kan, pMUXTp₁₂AB₅kan, and pMUXTp₁₃AB₅kan.

DETAILED DESCRIPTION OF THE INVENTION

[0021] By randomly inserting genes that encode PHA biosynthetic enzymesinto the chromosome of E. coli, means have been identified to directlyachieve high levels of expression from strong endogenous promoters atsites that are non-essential for growth of the host in industrial mediumbased fermentations. As demonstratd by the examples, E. coli strainshave been obtained using these techniques that produce PHAs in levelsexceeding 85% of the cell dry weight from single copy genes on thechromosome. Expression of the phb genes in these strains is notdependent on the upstream sequences of phbC in R. eutropha nor on a highcopy number construct. Maintenance of the phb genes by these strains isindependent of the supplementation of antibiotics, the presence ofstabilizing loci such as parB or hok/sok or any other selectivepressure. The ultra-high level of expression required in theplasmid-based systems has been found to be completely unnecessary.Furthermore, unlike the most successful fermentations reported to date(Wang & Lee, Biotechnol. Bioeng. 58:325-28 (1998)) for recombinantplasmid-based E. coli, fermentation with these strains provides thatvirtually all of the cells contain PHB at the end of the fermentation.

[0022] Despite the low copy number, these transgenic bacteria accumulatePHB to levels observed for wild-type organisms. The host used forrecombinant PHB production also is an important parameter in designing aplasmid-based E. coli system. For example, although W3110 strains werepoor PHB producers when using a plasmid-based system, it was found thatby integrating the phb genes into the chromosome of this same host, thehost retained excellent growth characteristics while accumulatingcommercially significant levels of PHB.

[0023] Methods and Materials for Producing the Microbial Strains

[0024] Bacterial Strains to be Modified

[0025] A number of bacteria can be genetically engineered to producepolyhydroxyalkanoates. These include organisms that already producepolyhydroxyalkanoates, modified to utilize alternative substrates orincorporate additional monomers, or to increase production, andorganisms that do not produce polyhydroxyalkanoates, but which expressesnone to some of the enzymes required for production ofpolyhydroxylkanoates. Examples include E. coli, Alcaligenes latus,Alcaligenese eutrophus, Azotobacter, Pseudomonas putida, and Ralstoniaeutropha.

[0026] Methods for Generating Transgenic PHB Producers

[0027] Methods for incorporating engineered gene constructs into thechromosomal DNA of bacterial cells are well known to those skilled inthe art. Typical integration mechanisms include homologous recombinationusing linearized DNA in recBC or recD strains followed by P1transduction (Miller 1992, A Short Course in Bacterial Genetics: Alaboratory manual & Handbook for Escherichia coli and Related Bacteria.Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y.) specialplasmids (Hamilton et al., J. Bacteriol. 171:4617 (1989); Metcalf etal., Plasmid 35:1 (1996); U.S. Pat. No. 5,470,727 to Mascarenhas etal.), or by random insertion using transposon based systems (Herrero etal. J. Bacteriol. 172:6557 (1990); Peredelchuk & Bennett, Gene 187:231(1997); U.S. Pat. No. 5,595,889 to Richaud et al.; U.S. Pat. No.5,102,797 to Tucker et al.). In general, the microbial strainscontaining an insertion are selected on the basis of an acquiredantibiotic resistance gene that is supplied by the integrated construct.However, complementation of auxotrophic mutants can also be used.

[0028] Expression of the genes of interest for chromosomal integrationcan be achieved by including a transcription activating sequence(promoter) in the DNA construct to be integrated. Site-directed,homologous recombination can be combined with amplification ofexpression of the genes of interest, as described by U.S. Pat. No.5,00,000 to Ingram et al. Although mini-transposon systems have beenused for a number of years, they have been designed such that theexpression level of the integrated gene of interest is not modulated.Ingram, et al. selected for increased expression of a foreign geneinserted into the E. coli chromosome by homologous recombination. Thiswas achieved by inserting a promoter-less chloroamphenicol (Cm)resistance gene downstream of the gene of interest to create atranscriptional fusion. After a transcriptional fusion of the alcoholdehydrogenase gene with a promoterless chloramphenicol acetyltransferase genes is integrated in the pfl gene, increased expression isachieved by selecting mutants on increasing concentrations ofchloramphenicol. However, in chemostat studies these stabilzed strainsstill lost the capacity to produce ethanol (Lawford & Rousseau, Appl.Biochem. Biotechnol. 57-58:293-305 (1996)). Also, strains that containedthe ethanologenic genes on the chromosome demonstrated a decreasedgrowth rate in glucose minimal medium (Lawford & Rousseau, Appl.Biochem. Biotechnol., 57-58:277-92 (1996)).

[0029] These approaches have been combined and modified to randomlyintegrate a mini-transposon into the chromosome to select for healthy,fast growing transgenic strains coupled with a screening system formodulating expression of the integrated genes. A series of expressioncassettes have been developed for inserting heterologous genes intobacterial chromosomes. These cassettes are based on the transposondelivery systems described by Herrero et al., J. Bacteriol. 172:6557-67(1990); de Lorenzo et al., J. Bacteriol. 172:6568 (1990). Although thesesystems specify RP4-mediated conjugal transfer and use only transposonTn10 and Tn5, any combination of transposon ends and delivery systemcould be adapted for the technology described, resulting in sustainedand homogeneous PHA production.

[0030] The following general approach is used for generating transgenicE. coli PHB producers: (1) a promoterless antibiotic resistance (abr)gene is cloned in the polylinker of a suitable plasmid such as pUC18NotIor pUC18SfiI so that the major part of the polylinker is upstream ofabr; (2) phb genes are subsequently cloned upstream of and in the sameorientation as the abr gene; (3) the phb-abr cassette is excised as aNotI or AvrII fragment (AvrII recognizes the SfiI site in pUC18SfiI) andcloned in the corresponding sites of any plasmid like those from thepUT- or pLOF-series; (4) the resulting plasmids are maintained in E.coli λpir strains and electroporated or conjugated into the E. colistrain of choice in which these plasmids do not replicate; and (5) newstrains in which the phb-abr cassette has successfully integrated in thechromosome are selected on selective medium for the host (e.g.,naladixic acid when the host is naladixic acid resistant) and for thecassette (e.g., chloramphenicol, kanamycin, tetracyclin, mercurychloride, bialaphos). The resulting phb integrants are screened onminimal medium in the presence of glucose for growth and PHB formation.

[0031] Several modifications of this procedure can be made. If thepromotorless antibiotic resistance marker is not used, the insertion ofthe PHA genes is selected based on a marker present in the vector andintegrated strains producing the desired level of PHA are detected byscreening for PHA production. The phb genes may have, but do not need,endogeneous transcription sequences, such as upstream activatingsequences, RNA polymerase binding site, and/or operator sequences. Ifthe phb genes do not have such sequences, the described approach islimited to the use of vectors like the pUT series in which transcriptioncan proceed through the insertion sequences. This limitation is due tothe inability of RNA polymerase to read through the Tn10 flankingregions of the pLOF plasmids. The abr gene may carry its own expressionsequences if so desired. Instead of an abr gene, the construct may bedesigned such that an essential gene serves as selective marker when thehost strain has a mutation in the corresponding wild-type gene. Examplesof genes useful for this purpose are generally known in the art.Different constructs can be integrated into one host, eithersubsequently or simultaneously, as long as both constructs carrydifferent marker genes. Using multiple integration events, phb genes canbe integrated separately, e.g., the PHB polymerase gene is integratedfirst as a phbC-cat cassette, followed by integration of the thiolaseand reductase genes as a phbAB-kan cassette. Alternatively, one cassettemay contain all phb genes whereas another cassette contains only somephb genes required to produce a desired PHA polymer.

[0032] In some cases a transposon integration vector such as pJMS11(Panke et al. Appl. Enviro. Microbiol. 64: 748-751) may be used suchthat the selectable marker can be excised from the chromosome of theintegrated strain. This is useful for a number of reasons includingproviding a mechanism to insert multiple transposon constructs using thesame marker gene by excising the marker following each insertion event.

[0033] Sources of phb and Other Genes Involved in PHA Formation

[0034] A general reference is Madison and Huisman, 1999, Microbiologyand Molecular Biology Reviews 63: 21-53. The phb genes may be derivedfrom different sources and combined in a single organism, or from thesame source.

[0035] Thiolase Encoding Genes

[0036] Thiolase encoding genes have been isolated from Alcaligeneslatus, Ralstonia eutropha (Peoples & Sinskey, J. Biol. Chem. 264(26):15298-303 (1989); Acinetobacter sp. (Schembri, et al., J. Bacteriol.177(15):4501-7 (1995)), Chromotium vinosum (Liebergesell & Steinbuchel,Eur. J. Biochem. 209(1%:135-50 (1992)), Pseudomonas acidophila,Pseudomonas denitrificans (Yabutani, et al., FEMS Microbiol. Lett. 133(1-2):85-90 (1995)), Rhizobium meliloti (Tombolini, et al., Microbiology141:2553-59 (1995)), Thiocystis violacea (Liebergesell & Steinbuchel,Appl. Microbiol. Biotechnol. 38(4):493-501 (1993)), and Zoogloearamigera (Peoples, et al., J. Biol. Chem. 262(1):97-102 (1987)).

[0037] Other genes that have not been implicated in PHA formation butwhich share significant homology with the phb genes and/or thecorresponding gene products may be used as well. Genes encodingthiolase- and reductase-like enzymes have been identified in a broadrange of non-PHB producing bacteria. E. coli (U29581, D90851, D90777),Haemophilus influenzae (U32761), Pseudomonas fragi (D10390), Pseudomonasaeruginosa (U88653), Clostridium acetobutylicum (U08465), Mycobacteriumleprae (U00014), Mycobacterium tuberculosis (Z73902), Helicobacterpylori (AE000582), Thermoanaerobacterium thermosaccharolyticum (Z92974),Archaeoglobus fulgidus (AE001021), Fusobacterium nucleatum (U37723),Acinetobacter calcoaceticus (L05770), Bacillus subtilis (D84432, Z99120,U29084), and Synechocystis sp. (D90910) all encode one or more thiolasesfrom their chromosome. Eukaryotic organisms such as Saccharomycescerevisiae (L20428), Schizosaccharomyces pombe (D89184), Candidatropicalis (D13470), Caenorhabditis elegans (U41105), human (S70154),rat (D13921), mouse (M35797), radish (X78116), pumpkin (D70895), andcucumber (X67696) also express proteins with significant homology to the3-ketothiolase from R. eutropha.

[0038] Reductase Encoding Genes

[0039] Reductase encoding genes have been isolated from A. latus, R.eutropha (Peoples & Sinskey, J. Biol. Chem. 264(26): 15298-303 (1989);Acinetobacter sp. (Schembri, et al., J. Bacteriol. 177(15):4501-7(1995)), C. vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem.209(1):135-50 (1992)), P. acidophila, P. denitrificans (Yabutani, etal., FEMS Microbiol. Lett. 133 (1-2):85-90 (1995)), R. meliloti(Tombolini, et al., Microbiology 141:2553-59 (1995)), and Z. ramigera(Peoples, et al., J. Biol. Chem. 262(1):97-102 (1987)).

[0040] Other genes that have not been implicated in PHA formation butwhich share significant homology with the phb genes and/or thecorresponding gene products may be used as well. Genes with significanthomology to the phbB gene encoding acetoacetyl CoA reductase have beenisolated from several organisms, including Azospirillum brasiliense(X64772, X52913) Rhizobium sp. (U53327, Y00604), E. coli (D90745),Vibrio harveyi (U39441), H. influenzae (U32701), B. subtilis (U59433),P. aeruginosa (U91631), Synechocystis sp. (D90907), H. pylori(AE000570), Arabidopsis thaliana (X64464), Cuphea lanceolata (X64566)and Mycobacterium smegmatis (U66800).

[0041] PHA Polymerase Encoding Genes

[0042] PHA polymerase encoding genes have been isolated from Aeromonascaviae (Fukui & Doi, J. Bacteriol. 179(15):4821-30 (1997)), A. latus, R.eutropha (Peoples & Sinskey, J. Biol. Chem. 264(26):15298-303 (1989);Acinetobacter (Schembri, et al., J. Bacteriol. 177(15):4501-7 (1995)),C. vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem. 209(1):135-50(1992)), Methylobacterium extorquens (Valentin & Steinbuchel, Appl.Microbiol. Biotechnol. 39(3):309-17 (1993)), Nocardia corallina (GenBankAce. No. AF019964), Nocardia salmonicolor, P. acidophila, P.denitrificans (Ueda, et al., J. Bacteriol. 178(3):774-79 (1996)),Pseudomonas aeruginosa (Timm & Steinbuchel, Eur. J. Biochem.209(1):15-30 (1992)), Pseudomonas oleovorans (Huisman, et al., J. Biol.Chem. 266:2191-98 (1991)), Rhizobium etli (Cevallos, et al., J.Bacteriol. 178(6): 1646-54 (1996)), R. meliloti (Tombolini, et al.,Microbiology 141 (Pt 10):2553-59 (1995)), Rhodococcus ruber (Pieper &Steinbuchel, FEMS Microbiol. Lett. 96(1):73-80 (1992)), Rhodospirrilumrubrum (Hustede, et al., FEMS Microbiol. Lett. 93:285-90 (1992)),Rhodobacter sphaeroides (Steinbuchel, et al., FEMS Microbiol. Rev.9(2-4):217-30 (1992); Hustede, et al., Biotechnol. Lett. 15:709-14(1993)), Synechocystis sp. (Kaneko, DNA Res. 3(3): 109-36 (1996)), T.violaceae (Liebergesell & Steinbuchel, Appl. Microbiol. Biotechnol.38(4:493-501 (1993)), and Z. ramigera (GenBank Acc. No. U66242).

[0043] Vectors for Incorporation of Genes into the Bacterial Chromosomes

[0044] The pUT and pLOF series of plasmid transposon delivery vectorsuseful in the PHA-producing methods described herein use thecharacteristics of transposon Tn5 and transposon Tn10, respectively. Thetransposase genes encoding the enzymes that facilitate transposition arepositioned outside of the transposase recognition sequences' and areconsequently lost upon transposition. Both Tn5 and Tn10 are known tointegrate randomly in the target genome, unlike, for example, the Tn7transposon. However, generally any transposon can be modified tofacilitate the insertion of heterologous genes, such as the phb genes,into bacterial genomes. This methodology thus is not restricted to thevectors used in the methods described herein.

[0045] Methods and Materials for Screening for Enhanced PolymerProduction

[0046] Screening of Bacterial Strains

[0047] The technology described above allows for the generation of newPHA producing strains and also provides new bacterial strains that areuseful for screening purposes. Table 1 below shows the differentcombinations of chromosomally and plasmid encoded PHB enzymes and howspecific strains can be used to identify new or improved enzymes.

[0048] Besides a screening tool for genes that express improved enzymes,E. coli strains with a complete PHA pathway integrated on the chromosomecan be used to screen for heterologous genes that affect PHA formation.E. coli is a useful host because genes are easily expressed from amultitude of plasmid vectors: high copy-number, low copy-number,chemical or heat inducible, etc. and mutagenesis procedures have beenwell established for this bacterium. In addition, the completelydetermined genomic sequence of E. coli facilitates the characterizationof genes that affect PHA metabolism.

[0049] Transgenic E. coli strains expressing an incomplete PHA pathwaycan be transformed with gene libraries to identify homologs of themissing gene from other organisms, either prokaryotic or eukaryotic.Because these screening strains do not have the complete PHAbiosynthetic pathway, the missing functions can be complemented andidentified by the ability of the host strain to synthesize PHA.Generally PHA synthesizing bacterial colonies are opaque on agar plates,whereas colonies that do not synthesize PHA appear translucent. Clonesfrom a gene library that complement the missing gene confer a whitephenotype to the host when grown on screening media. Generally screeningmedia contains all essential nutrients with excess carbon source and anantibiotic for which resistance is specified by the vector used in thelibrary construction.

[0050] Besides new genes, genes encoding improved PHA biosyntheticenzymes can also be screened for. A mutagenized collection of plasmidscontaining a phb biosynthetic gene into an E. coli host strain lackingthis activity but containing genes encoding the other PHA biosyntheticenzymes can be screened for increased or altered activity. For example,PHA polymerases with increased activity can be screened for in a strainthat expresses thiolase and reductase from the chromosome by identifyingPHB-containing colonies under conditions that support PHB formationpoorly. mcl-PHA polymerases with an increased specificity towards C₄ cansimilarly be screened for under PHB accumulation promoting conditions.Altered activities in the phaG encoded ACP::CoA transferase can bescreened for by expressing mutated versions of this gene in a phbCintegrant and screening for PHB formation from short chain fatty acids.Enzymes that have increased activity under sub-optimal physicalconditions (e.g., temperature, pH, osmolarity, and oxygen tension) canbe screened for by growing the host under such conditions and supplyinga collection of mutated versions of the desired gene on a plasmid.Reductase enzymes with specificity to medium side-chain3-ketoacyl-CoA's, such as 3-ketohexanoyl-CoA, can be screened for byidentifying PHA synthesizing colonies in a strain that has a msc-PHApolymerase gene integrated on the chromosome and mutagenized versions ofa phbB gene on a plasmid. The combination of different specificity PHAenzymes allows for the screening of a multitude of new substratespecificities. Further permutations of growth conditions allows forscreening of enzymes active under sub-optimal conditions or enzymes thatare less inhibited by cellular cofactors, such as Coenzyme A andCoA-derivatives, reduced or oxidised nicotinamide adenine dinucleotideor nicotinamide adenine dinucleotide phosphate (NAD, NADP, NADH, andNADPH).

[0051] Using the techniques described herein, E. coli strains expressingthe genes encoding enzymes for the medium side-chain PHA pathway can beconstructed. Strains in which either phaC or phaG or both are integratedon the chromosome of E. coli accumulate a PHA including mediumchain-length 3-hydroxy fatty acids of which 3-hydroxydecanoate is thepredominant constituent. When phaC is integrated by itself, msc-PHAs canbe synthesized from fatty acids. In such strains, it is advantageous tomanipulate fatty acid oxidation such that 3-hydroxy fatty acidprecursors accumulate intracellularly. This manipulation can be achievedby mutagenesis or by substituting the E. coli fatty acid degradationenzymes FadA and FadB encoding genes with the corresponding faoAB genesfrom Pseudomonas putida or related rRNA homogy group I fluorescentpseudomonad. TABLE 1 Phenotypes of Strains for Screening of New orImproved Enzymes Genes integrated Gene(s) on Carbon source Screenidentifies on chromosome plasmid for screen genes encoding phbC libraryglucose new thiolase/ reductase library fatty acids new reductase,hydratase, trans- ferase library hydroxy fatty acid, hydroxy e.g. 4-fatty acid hydroxybutyrate activating (4HB) enzyme, e.g. 4HB-CoA trans-ferase (acetyl- CoA or succinyl- CoA dependent) or 4HB-CoA synthase phaGglucose transferase with new substrate specificity phbAB library glucosenew polymerase gene phaC glucose; altered polymerase with environmentalnew substrate conditions specificity; increased activity undersub-optimal conditions phbBC library glucose new thiolase phbA limitingglucose/less deregulated prefered carbon thiolase; sources or richincreased medium; altered activity under environmental sub-optimalconditions conditions phbAC library glucose new reductase phbB limitingglucose/less deregulated prefered carbon reductase; sources or richincreased activity medium; altered under sub-optimal environmentalconditions conditions phbCAB library any enzymes affecting PHB formationunder specific conditions phbCAB, random any enzymes affecting mutationsPHB formation (chemical or under specific transposon) conditions phaClibrary hexanoate hydratase with specificity for C6 and longersubstrates phaJ fatty acids hydratase with increased specificity for C6and longer substrates phbB fatty acids reductase with new substratespecificity phaC fadR⁺, Δato phbAB glucose + butyrate thiolase/reductasecombination specific for C6 monomer phaJ phaC fatty acids polymerasewith wider substrate specificity phaG phbC glucose polymerase with widersubstrate specificity

EXAMPLES

[0052] The methods and compositions described herein will be furtherunderstood by reference to the following non-limiting examples. Theseexamples use the following general methods and materials.

[0053] Materials and Methods

[0054]E. coli strains were grown in Luria-Bertani medium (Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1992 at 37° C. or 30° C. orin minimal E2 medium (Lageveen et al., Appl. Environ. Microbiol 54:2924-2932 (1988)). DNA manipulations were performed on plasmid andchromosomal DNA purified with the Qiagen plasmid preparation or Qiagenchromosomal DNA preparation kits according to manufacturersrecommendations. DNA was digested using restriction enzymes (New EnglandBiolabs, Beverly, Mass.) according to manufacturers recommendations. DNAfragments were isolated from 0.7% agarose-Tris/acetate/EDTA gels using aQiagen kit.

[0055] Plasmid DNA was introduced into E. coli cells by transformationor electroporation (Sambrook, et al., Molecular Cloning: A LaboratoryManual, 2d ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.)). Transposition of phb genes from the pUT vectors was achieved bymating of the plasmid donor strain and the recipient (Herrero et al., J.Bacteriol. 172:6557 (1990)). The recipient strains used were spontaneousnaladixic acid or rifampicin resistant mutants of E. coli derived fromeither LS5218 or MBX23. MBX23 is LJ14 rpoS::Tn10 in which the rpoS::Tn10allele was introduced by P1 transduction from strain 1106 (Eisenstark).Recipients in which phb genes have been integrated into the chromosomewere selected on naladixic acid or rifampicin plates supplemented withthe antibiotic resistance specified by the mini-transposon, kanamycin,or chloramphenicol. Oligonucleotides were purchased from Biosynthesis orGenesys. DNA sequences were determined by automated sequencing using aPerkin-Elmer ABI 373A sequencing machine. DNA was amplified using thepolymerase-chain-reaction in 50 microliter volume using PCR-mix fromGibco-BRL (Gaithersburg, Md.) and an Ericomp DNA amplifying machine.

[0056] DNA fragments were separated on 0.7% agarose/TAE gels. Southernblots were performed according to procedures described by Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Detection of DNA fragmentscontaining phb genes was performed using chemiluminescent labeling anddetection kits from USB/Amersham. Proteins samples were denatured byincubation in a boiling water bath for 3 minutes in the presence of2-mercaptoethanol and sodium dodecylsulphate and subsequently separatedon 10%, 15%, or 10-20% sodium dodecylsulphate-polyacrylamide gels. Aftertransfer of protein to supported nitrocellulose membranes (Gibco-BRL,Gaithersburg, Md.), 3-ketoacyl-CoA thiolase, acetoacetyl-CoA reductaseand PHB polymerase was detected using polyclonal antibodies raisedagainst these enzymes and horseradish peroxidase labeled secondaryantibodies followed by chemiluminescent detection (USB/Amersham).

[0057] Acetoacetyl-CoA thiolase and acetoacetyl-CoA reductase activitieswere determined as described by Peoples and Sinskey, J. Biol. Chem. 264:15293-15297 (1989) in cell free extracts from strains grown for 16 hoursin LB-medium at 37 C. The acetoacetyl-CoA thiolase activity is measuredas degradation of a Mg²⁺-acetoacetyl-CoA complex by monitoring thedecrease in absorbance at 304 m after addition of cell-free extractusing a Hewlett-Packer spectrophotometer. The acetoacetyl-CoA reductaseactivity is measured by monitoring the conversion of NADH to NAD at 340nm using a Hewlett-Packer spectrophotometer.

[0058] Accumulated PHA was determined by gas chromatographic (GC)analysis as follows. About 20 mg of lyophilized cell mass was subjectedto simultaneous extraction and butanolysis at 110 C for 3 hours in 2 mLof a mixture containing, by volume, 90% 1-butanol and 10% concentratedhydrochloric acid, with 2 mg/mL benzoic acid added as an internalstandard. The water-soluble components of the resulting mixture wereremoved by extraction with 3 mL water. The organic phase (1 μL at asplit ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed onan HP 5890 GC with FID detector (Hewlett-Packard Co, Palo Alto, Calif.)using an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25μm film; Supelco; Bellefonte, Pa.) with the following temperatureprofile: 80° C., 2 min.; 10° C. per min. to 250° C.; 250° C., 2 min. Thestandard used to test for the presence of 4-hydroxybutyrate units in thepolymer was γ-butyrolactone, which, like poly(4-hydroxybutyrate), formsn-butyl 4-hydroxybutyrate upon butanolysis. The standard used to testfor 3-hydroxybutyrate units in the polymer was purified PHB.

[0059] The molecular weights of the polymers were determined followingchloroform extraction by gel permeation chromatography (GPC) using aWaters Styragel HT6E column (Millipore Corp., Waters ChromatographyDivision, Milford, Mass.) calibrated versus polystyrene samples ofnarrow polydispersity. Samples were dissolved in chloroform at 1 mg/mL,and 50 μL samples were injected and eluted at 1 mL/min. Detection wasperformed using a differential refractometer.

[0060] 1-Methyl-3-nitro-1-nitroso-guanidine (NTG) mutagenesis wasperformed as described by Miller, A Short Course in Bacterial Genetics(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using a90 minute treatment with 1 mg/ml NTG corresponding to 99% killing.

Example 1 Host Strains and Plasmid Tools for Gene Integration

[0061] Strains and plasmids from which transposon vectors and transposonderivatives were developed are listed in Tables 2 and 3 below. MBX245and MBX247 were selected by growing MBX23 and LS5218 respectively on LBplates containing approximately 30 g/ml naladixic acid. MBX246 andMBX248 were selected by growing MBX23 and LS5218, respectively, on LBplates containing 50 g/ml rifampicin. Colonies that appeared on theseselective media within 24 hours were replica plated on the same mediumand after growth stored in 15% glycerol/nutrient broth at −80° C.

[0062] MBX245 and MBX247 were selected by growing MBX23 and LS5218respectively on LB plates containing 30 μg/ml naladixic acid. MBX246 andMBX248 were selected by growing MBX23 and LS5218 respectively on LBplates containing 50 μg/ml rifampicin. Colonies that appeared on theseselective media within 24 hours were replica plated on the same mediumand after growth stored in 15% glycerol/nutrient broth at −80° C. TABLE2 Host Strains Used For Gene Integration strain genotype source DH5αrecA1 endA1 gyrA96 thi 1 hsdR17 supE44 relA1 Δ(lac- proAB)(Φ80dlacΔ(lacZ)M15) S17-1 λpir recA thi pro hsdR⁻M⁺ RP4:2- 2 Tc::Mu::KmTn7 λpir lysogen CC118 λpir Δ(ara-leu) araD ΔlacX74 galE 2 galK phoA20thi-1 rpsE rpoB argE(Am) recA1, λpir lysogen XL1-Blue F′::Tn10 lacI^(q)Δ(lacZ) M15 3 proAB/recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 Δ(lac-proAB) LS5218 fadR601 atoC512^(c) Spratt et al, 1981 J. Bacteriol. 146:1166-1169 LJ14 LS5218 atoC2^(c) atoA14 Spratt et al, 1981 J. Bacteriol.146: 1166-1169 MBX23 LJ14 rpoS Metabolix, Inc. MBX245 MBX23 Nl^(r)Metabolix, Inc. MBX246 MBX23 Rf^(r) Metabolix, Inc. MBX247 LS5218 Nl^(r)Metabolix, Inc. MBX248 LS5218 Rf^(r) Metabolix, Inc.

[0063] TABLE 3 Plasmids Used For Gene Integration plasmidcharacteristics source pUC18Not Ap^(r), NotI sites flanking polylinker 2pUC18Sfi Ap^(r), SfiI sites flanking polylinker 2 pUTkan Ap^(r), Km^(r),oriR6K, mobRP4 2 depends on λpir for replication pUTHg Ap^(r), Hg^(r),oriR6K, mobRP4 2 depends on λpir for replication pKPS4 Ap^(r), phaC1from Pseudomonas oleovorans pUCDBK1 Ap^(r), phbA and phbB from Peoplesand Sinskey Zoogloea ramigera 1989, Molecular Microbiol.3: 349-357 pZSAp^(r), phbC from WO 99/14313 Zoogloea ramigera

Example 2 Construction of Cloning Vectors to Facilitate Integration ofphb Genes

[0064] The plasmids pMNXTp1kan and pMNXTp1cat were based on the plasmidspUC18Not and pUC18Sfi and developed as shown in FIG. 1.

[0065] The Tn903 kanamycin (Km) resistance gene from plasmid pBGS18 wasamplified by PCR using the oligonucleotide primers linkK1,

[0066] 5′ TGCATGCGATATCAATTGTCCA GCCAGAAAGTGAGG,

[0067] and linkK2,

[0068] 5′ ATTTATTCAACAAAGCCGCC.

[0069] Prior to PCR amplification, the primers were phosphorylated usingT4 polynucleotide kinase using standard procedures. The DNa wasamplified using the following program: 1 cycle of 3 min at 95° C., 40 sat 42° C., 2 min at 72° C., followed by 30 cycles of 40 s at 95° C., 40s at 42° C. and 90 s at 72° C. The DNA then was phenol extracted andtreated with T4 DNA polymerase prior to gel purification. The bluntended 0.8 kb DNA fragment was then inserted into the Ecl136II site inthe polylinker of pUC18Not to obtain pMNXkan.

[0070] The cat gene was obtained as an HindIII cassette from Pharmacia(Pharmacia Inc. NJ), blunt ended using Klenow fragment of DNApolymerase, and inserted into the Ecl136II site of pUC18Not to obtainpMNXcat.

[0071] The trp terminator sequence was constructed by annealing the twosynthetic oligonucleotides TERM1

[0072] (5′ CCCAGCCCGCTAATGAGCGGGCTTTTTTTTGAACAA AA 3′)

[0073] and TERM2

[0074] (5′ TACGTATTTTGTTCAAAAAAAAGCCCGCTCATTAGCGGG CTGGG 3′).

[0075] The terminator was then inserted into the HindIII-SphI site ofpMNXkan and pMNXcat to obtain pMNXTkan and pMNXTcat, respectively. Thesevectors were constructed such that any promoter fragment can be addedbetween the SphI and Sac sites. Promoter p, was constructed by annealingof the synthetic oligonucleotides PHBB1

[0076] (5′ TACGTACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGT GTGGAATTGTGAGCGGTT 3′)

[0077] and PHBB2

[0078] (5′ TTCGAACCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAATGTAAAGCCTGGGG3′)

[0079] followed by filling in the ends with Klenow fragment of DNApolymerase. The blunt-ended promoter fragment p, was then inserted intothe HincII site of pMNXTkan and pMNXTcat to obtain pMNXTp₁kan andpMNXTp₁cat, respectively.

[0080] Plasmid pMSXTp₁cat was constructed by transferring the Tp₁catcassette from pMNXTp₁cat as an EcoRI-HindIII fragment into theEcoRI-HindIII site of pUC18Sfi. Similarly, pMSXTp₁kan was constructed bytransferring the EcoRI-HindIII fragment containing Tp₁kan into theEcoRI-HindIII site of pUC18Sfi.

Example 3 Construction of Plasmids for Chromosomal Integration of phbC,Encoding PHB Polymerase

[0081] Plasmid pMUXC₅cat contains the phbC gene from Z. ramigera on atransposable element for integration of this gene on the chromosome of arecipient strain, as shown in FIG. 2. Strong translational sequenceswere obtained from pKPS4 which includes phaC1 encoding PHA polymerasefrom P. oleovorans in the pTrc vector (Pharmacia). In this construct,phaC1 is preceded by a strong ribosome binding site: AGGAGGTTTTT(-ATG).The phaC1 gene including the upstream sequences, was cloned as a bluntended EcoRI-HindIII fragment in the SmaI site of pUC18Sfi to givepMSXC₃. A blunt ended cat gene cassette was subsequently cloned in theblunt-ended Sse8387II site, resulting in pMSXC₃cat. At this point, allof the phaC1 coding region except the 5′ 27 base pairs were removed as aPstI-BamHI fragment and replaced by the corresponding fragment from thephbC gene from Z. ramigera. The resulting plasmid pMSXC₃cat encodes ahybrid PHB polymerase enzyme with the 9 amino terminal residues derivedfrom the P. oleovorans PHA polymerase and the remainder from Z.ramigera. The C₅cat cassette was then excised as an AvrII fragment andcloned in the corresponding sites of pUTHg, thereby deleting the mercuryresistance marker from this vector. The resulting plasmid, pMUXC₅cat,contains a C₅cat mini-transposon in which phbC is not preceded by apromoter sequence. Expression of the cassette upon integration istherefore dependent on transcriptional sequences that are provided bythe DNA adjacent to the integration site.

Example 4 Construction of Plasmids for Chromosomal Integration of phbAB,Encoding Thiolase and Reductase

[0082] pMSXTp₁AB₅kan2 was constructed from pMSXTp₁kan as partially shownin FIG. 3. First pMSXTp₁kan was digested with NdeI, filled in withKlenow and religated to obtain pMSXTp₁kan2 in which the NdeI site isdeleted. This deletion results in a unique NdeI site just upstream ofphbA of Z. ramigera during later stages of the cloning procedure.

[0083] B₅ was cloned as a NarI fragment from pUCDBK1 (Peoples andSinskey 1989, Molecular Microbiol. 3: 349-357) and cloned in the HincIIsite of pUC18Sfi to generate pMSXB₅. A₅ was inserted as anFseI/blunt-SalI fragment in the Ecl36II-SalI sites resulting in pMSXAB₅and regenerating the Z. ramigera AB₅ intergenic region. pMSXAB₅cat wascreated by inserting a promoterless cat cassette in the HindIII site ofpMSXAB₅. The AB₅ fragment from pMSXAB₅cat was cloned as a EcoRI-PstIfragment into the SmaI site of pMSXTp₁kan2 giving pMSXTp₁AB₅kan2.

[0084] The expression cassette AB₅cat was then excised as a 2.8 kb AvrIIfragment and ligated into the AvrII site of pUTHg and transformed intoE. coli strain CC118 λpir to obtain plasmid pMUXAB₅cat. This plasmid wasthen transformed into E. coli S17-1λpir and used to insert the AB5catexpression cassette into the chromosome of E. coli MBX247 byconjugation. The resulting Ap^(s)/Cm^(r) transconjugants werecharacterized for integration and expression of the thiolase andreductase genes encoded by the phbAB genes.

Example 5 Construction of Plasmids with Improved Promoters forIntegration of phbAB into the Chromosome of E. coli

[0085] Expression of phbAB5 was improved by introduction of strongpromoters upstream of these genes, as shown in FIG. 3. These promoterswere generated with sets of oligonucleotides that provide upstreamactivating sequences, a −35 promoter region, a −10 promoter region withtranscriptional start site(s), and mRNA sequences with possiblestabilizing functions. Plasmid pMSXTp₁AB₅kan2 was digested withPstI/XbaI, and a fragment containing the −10 region of the lac promoterwas inserted as a fragment obtained after annealing oligonucleotides 3A

[0086] (5′ GGCTCGTATAATGTGTGGAGGGAGAACCGCCGGGCTCGCGCCGTT)

[0087] and 3B

[0088] (5′ CTAGAACGGCGCGAGCCCGGCGGTTCTCCCTCCACACATTATAC GAGCCTGCA).

[0089] Next, a fragment containing the lac −35 region and the rrnBregion were inserted into the PstI site as a fragment obtained afterannealing the oligonucleotides: 1A(5′ TTCAGAAAATTATTTTAAATTTCCTCTTGACATTTATGCT GCA) and 1B(5′ GCATAAATGTCAAGAGGAAATTTAAAATAATTTTCTGAATGCA).

[0090] Next, the messenger stabilizing sequence including thetranscriptional start site from AB₅ was inserted into the XbaI-NdeIsites as a fragment obtained after annealing the oligonucleotides: 4A(5′ CTAGTGCCGGACCCGGTTCCAAGGCCGGCCGCAAGGCTGCCAGAAC TGAGGAAGCACA) and 4B(5′ TATGTGCTTCCTCAGTTCTGGCAGCCTTGCGGCCGGCCTTGGAACC GGGTCCGGCA).

[0091] The resulting plasmid is pMSXp₁₁AB₅kan2. The AvrII fragment,containing Tp₁₁AB₅kan2 was cloned into pUTHg cut with AvrII and used forintegration into the genome of MBX379 and MBX245.

[0092] Plasmid pMSXTp₁₂AB₅kan2 was constructed as pMSXTP₁₁AB₅kan2 withthe distinction that the following oligonucleotides were used instead ofoligonucleotides 1A and 1B: 2A: (5′ TCCCCTGTCATAAAGTTGTCACTGCA) and 2B(5′ GTGACAACTTTATGACAG GGGATGCA).

[0093] These oligonucleotides provide a consensus E. coli pho box and−35 promoter region to generate a promoter that is potentially regulatedby the phosphate concentration in the medium.

[0094] pMSXTp₁₃AB₅kan2 was constructed to provide expression of AB₅ froma promoter that has been shown to be expressed under general stressconditions such as nutrient limitation, pH or heat shock, andadministration of toxic chemicals. The promoter region of uspA wasamplified using oligonucleotides UspUp (5′ TGACCAACATACGA GCGGC) andUspDwn (5′ CTACCAGAACTTTGCTTTCC)

[0095] in a PCR reaction consisting of an incubation at 95 C for 3 min.followed by 30 cycles of 40 s at 95° C., 40 s at 42° C., an incubationfor 7 min. at 68° C., and final storage at 4° C. The approximately 350bp PCR product was cloned into pCR2.1 (Invitrogen Corp., USA) togenerate pMBXp₁₃. An approximately 190 bp HincII-MscI fragmentcontaining the promoter and transcriptional start site for uspA and thefirst 93 bp of the uspA mRNA was cloned into blunt ended BamHI-Sse8387IpMSXTp₁kan2 to give pMSXTp₁₃kan2. Plasmid pMSXTp₁₃kan2 was then KpnIdigested, blunt ended with T4 polymerase and dephosphorylated using calfintestinal phosphatase. The AB₅ genes were isolated as a 2.0 kbEcoRI/Sse8387I fragment from pMSXAB₅, blunt ended using Klenow and T4polymerase and ligated into the KpnI site of pMSXTp₁₃kan2. In theresulting plasmid pMSXTp₁₃AB₅kan2, the phbAB and kan genes are expressedfrom the uspA (p₁₃) promoter.

[0096] The p_(n)AB₅kan (n=11, 12, 13) expression cassettes were thenexcised as 2.8 kb AvrII fragments and ligated into the AvrII site ofpUTHg and transformed into E. coli strain CC118 λpir to obtain plasmidpMUXp_(n)AB₅kan. This plasmid was then transformed into E. coliS17-1λpir and used to insert p₁₁AB₅kan, p₁₂AB₅kan, and p₁₃AB₅kanexpression cassettes into the chromosome of E. coli strains byconjugation.

Example 6 Integration of C₅cat into the Chromosome of E. coli

[0097] C₅cat was introduced into the chromosome of MBX23 by conjugationusing S17-1 λpir (pMUXC₅cat) as the donor strain. The conjugationmixture was spread on LB/N1/Cm plates and integrants were obtained, 40%of which were sensitive to ampicillin, indicating that no plasmid waspresent in these strains. Five integrants were transformed withpMSXAB₅cat (Ap^(r)) and grown on LB/Ap/Cm/2% glucose to examinebiosynthetic activity of PHB polymerase (Table 4). TABLE 4 IntegratedStrains strain containing strain after strain pMSXAB5cat PHB phenotypeplasmid curing MBX300 MBX305 ++++ MBX325 MBX301 MBX308 +++ MBX331 MBX302MBX310 ++++ MBX326 MBX303 MBX315 ++++ MBX327 MBX304 MBX316 + MBX337

Example 7 Amplification of C5 Expression in Integrated Strains

[0098] Expression of PHB polymerase was increased by restreaking MBX326successively on LB plates containing 100, 200, 500, and 1000 μg/mlchloroamphenicol. Strain MBX379 was derived from MBX326 and exhibitedchloramphenicol resistance up to 1000 μg/ml. In Southern blot analysisof chromosomal DNA isolated from MBX379 and its predecessors, the phbC5copy-number had not increased. Western blot analysis indicated a strongincrease in PHB polymerase levels in cell free extracts of these strainswhen the phbAB genes were present on a plasmid.

Example 8 Integration of p₁₁AB₅kan, p₁₂AB₅kan and p₁₃AB₅kan into MBX379

[0099] S17-1 λpir strains with either pMUXp₁₁AB₅kan, pMUXp₁₂AB₅kan, orpMUXp₁₃AB₅kan were mated with MBX379. Transgenic strains in whichphbAB₅kan had integrated on the chromosome were selected on LB/N1/Kmplates. Among the integrants, PHB producers were identified onLB/glucose plates. Representatives of the individual constructs wereMBX612 (MBX379::p₁₁AB₅kan), MBX677 (MBX379:: p₁₂AB₅kan), and MBX680(MBX379::p₁₃AB₅kan). Southern blots and Western blots showed that thephbAB genes had integrated in the chromosome and were expressed in thesestrains as well. Table 5 shows the PHB accumulation levels of transgenicE. coli PHB producers grown in Luria-Bertani medium with 2% glucose orminimal E2 medium with 2% glucose and 0.5% corn steep liquor. TABLE 5PHB Accumulation Levels for Transgenic E. coli PUB Producers % PHB ofcell dry weight strain LB/glucose E2 glucose MBX612 56 35 MBX677 58 38MBX680 39 50

Example 9 Selection and Bacteriophage P1 Transduction to Yield ImprovedStrains

[0100] The growth characteristics of MBX612, 677, and 680 were improvedby bacteriophage P1 transduction. A single transduction step wasrequired to transduce the C₅cat and AB₅kan alleles from the differentstrains into LS5218, indicating that the two separate integrationcassettes were located close to each other on the chromosome. Theresulting strains are MBX690 (from MBX681), MBX691 (from MBX677), andMBX698 (from MBX680). Repeated inoculation of MBX612 on minimal E2medium with limiting nitrogen resulted in MBX681. Unlike the strainsgenerated by P1 transduction, MBX681 did not exhibit improved growthcharacteristics. Southern blots and Western blots show that phbC and thephbAB genes were successfully transduced and were expressed in thesestrains as well. Table 6 below shows PHB accumulation levels for thesetransgenic E. coli PHB producers grown in Luria-Bertani medium with 2%glucose or minimal E2 medium with 2% glucose and 0.5% corn steep liquor.TABLE 6 PHB Accumulation Levels for Transgenic E. coli PHB Producers %PHB of cell dry weight strain LB/glucose E2 glucose MBX681 54 22 MBX69052 44 MBX691 54 28 MBX698 37 15

Example 10 Further Improvements of Transgenic E. coli Strains for PHBProduction

[0101] Mutagenesis using NTG or EMS was used to further improve PHBproduction in MBX680. Strains MBX769 and MBX777 were selected aftertreatment of MBX680 with EMS and NTG, respectively. These strains werefound to be able to grow on R2-medium supplied with 1% glucose, 0.5%corn steep liquor, and 1 mg/ml chloroamphenicol. MBX769 was grown in 50ml R-10 medium/0.5% CSL with 2 or 3% glucose at 37° C. for 20 to 26hours. PHB was accumulated to 71% of the cell dry weight. Similarly,MBX769 was grown in 50 ml LB with or without 0.375 g/L KH₂PO₄, 0.875K₂HPO₄, 0.25 (NH₄)₂SO₄, and a total of 50 g/L glucose (five aliquotswere added over the course of the incubation). After 63 hours ofincubation, PHB had accumulated up to 96% of the cell dry weight.

[0102] The phbC and phbAB alleles from MBX777 were subsequentlytransduced into LS5218, resulting in MBX820. Southern blots and Westernblots show that phbC and the phbAB genes were successfully transducedand were expressed in these strains as well. Table 7 shows the PHBaccumulation levels of these transgenic E. coli PHB producers grown inLuria-Bertani medium with 2% glucose or minimal E2 medium with 2%glucose and 0.5% corn steep liquor. TABLE 7 PHB Accumulation Levels forTransgenic E. coli PHB Producers % PHB of cell dry weight strainLB/glucose E2 glucose MBX680 39 50 MBX777 67 57 MBX820 53 50

Example 11 Growth Characteristics of Transgenic E. coli PHB Producers

[0103] The introduction of phb genes into MBX245 (t_(d)=47 min.) wasaccompanied by a reduction in growth rate (MBX680, t_(d)=71 min.).Improved PHB production was achieved by EMS mutagenesis, but did notimprove the growth rate (MBX777, t_(d)=72 min.). P1 transduction of thePHB genes into a wild-type strain (MBX184) resulted in the same highgrowth rate as exhibited by MBX245 and PHB accumulation up to 50% of thecell dry weight in less than 24 hours (MBX820, t_(d)=45 min.).

Example 12 Plasmids for Chromosomal Integration of Other pha Genes

[0104] The integration of phbC, phbA, and phbB from Z. ramigeradescribed herein also is applicable to other pha genes, such as genesencoding PHB polymerase from R. eutropha (C₁), PHA polymerase from P.oleovorans (C3), PHB polymerase from A. caviae (C 12), ACP::CoAtransacylase from P. putida (G3), (R)-specific enouyl-CoA hydratase fromA. caviae (J12), a broad substrate specific 3-ketoacyl-CoA thiolase fromR. eutropha (A1-II), or a phasin from R. eutropha (P1-I and P1-II).These genes were obtained by polymerase chain reaction amplificationusing the following primers: C1 up 5′ g-GAATTC-aggaggtttt-ATGGCGACCGGCAAAGGCGCGGCAG 3′ C1 dw5′ GC-TCTAGA-AGCTT-tcatgccttggctttgacgtatcgc 3′ C3 up5′ g-GAATTC-aggaggtttt- ATGAGTAACAAGAACAACGATGAGC 3′ C3 dw5′ GC-TCTAGA-AGCTT-tcaacgctcgtgaacgtaggtgccc 3′. C12 up5′ g-GAATTC-aggaggtttt- ATGAGCCAACCATCTTATGGCCCGC 3′ C12 dw5′ GC-TCTAGA-AGCTT-TCATGCGGCGTCCTCCTCTGTTGGG 3′ G3 up5′ g-GAATTC-aggaggtttt- ATGAGGCCAGAAATCGCTGTACTTG 3′ G3 dw5′ GC-TCTAGA-AGCTT-tcagatggcaaatgcatgctgcccc 3′ J12 up5′ ag-GAGCTC-aggaggtttt- ATGAGCGCACAATCCCTGGAAGTAG 3′ J12 dw5′ GC-TCTAGA-AGCTT-ttaaggcagcttgaccacggcttcc 3′ A1-II up5′ g-GAATTC-aggaggtttt- ATGACGCGTGAAGTGGTAGTGGTAAG 3′ A1-II dw5′ GC-TCTAGA-AGCTT-tcagatacgctcgaagatggcggc 3′. P1-I up5′ g-GAATTC-aggaggtttt- ATGATCCTCACCCCGGAACAAGTTG 3′ P1-I dw5′ GC-TCTAGA-AGCTT-tcagggcactaccttcatcgttggc 3′ P1-II up5′ g-GAATTC-aggaggtttt- ATGATCCTCACCCCGGAACAAGTTG 3′ P1-II dw5′ GC-TCTAGA-AGCTT-tcaggcagccgtcgtcttctttgcc 3′

[0105] PCR reactions included 10 pmol of each primer, 1 to 5 μl ofchromosomal DNA or boiled cells, and 45 μl PCR mix from Gibco BRL(Gaithersburg, Md.). Amplification was by 30 cycles of 60 s incubationat 94 C, 60 s incubation at a temperature between 45 C and 68 C and 1 to3 minutes incubation at 72 C. PCR products were purified, digested withEcoRI and HindIII, blunt ended with the Klenow fragment of DNApolymerase, and cloned in the SmaI site of pMSXcat, pMSXkan, pMNXcat, orpMNXkan according to the schemes shown in FIGS. 1 and 2. pMUXpha wasderived from pUTHg or pUTkan; and pMLXpha was derived from pLOFHg, wherepha stands for the pha gene of choice. These plasmids were used forintegration of the desired pha gene into the chromosome of E. coli orany other Gram-negative microbial strain suitable for PHA production

Example 13 PHBV Copolymer Producing Transgenic E. coli Strains

[0106]E. coli strains with chromosomally integrated phb genes such asdescribed above also can be used to produce PHBV copolymers. PHBV isgenerally synthesized in fermentation systems where propionic acid isco-fed with glucose or other carbohydrate. After uptake, propionate isconverted to propionyl-CoA, which by the action of acyl-CoA thiolase and3-ketoacyl-CoA reductase is converted to 3-hydroxyvaleryl-CoA (3HV-CoA).3HV-CoA is subsequently polymerized by PHA polymerase.

[0107] The capacity to accumulate PHBV can be increased by increasinglevels of enzymes that specifically synthesize HV monomers. Such enzymesmay be involved in the uptake of propionic acid, in the activation ofpropionic acid to propionyl-CoA or in any of the PHB biosyntheticenzymes. Additionally, alternative enzymes can be isolated from othersources, or propionyl-CoA can be obtained from alternative pathways,e.g. from the methylmalonyl-CoA pathway. In this pathway, succinyl-CoAis converted to methylmalonyl-CoA which is then decarboxylated to yieldpropionyl-CoA.

Example 14 PHB-4HB Copolymer Producing Transgenic E. coli Strains

[0108] Homopolymers and copolymers containing 4HB monomers can beproduced by transgenic E. coli strains. Incorporation of 4HB from4HB-CoA can be achieved by feeding 4-hydroxybutyrate to the PHAproducing organisms. 4HB is activated to 4HB-CoA either through a4-hydroxybutyryl-CoA transferase such as hbcT (OrfZ) from Clostridiumkluyveri or by an endogenous E. coli enzyme or by any other enzyme withthis capability. A P4HB homopolymer is produced when the transgenic E.coli strain contains only the phbC gene. 4HB containing copolymers canbe synthesized when the transgenic E. coli strain contains genesencoding the complete PHB biosynthetic pathway.

[0109]E. coli MBX821 (LS5218::C₅-cat³⁷⁹, atoC^(c)) was grown inLuria-Bertani medium and resuspended in 100 ml 10% LB with 5 g/L 4HB and2 g/L glucose. After incubation of this culture for 24 hours, PHA wascharacterized and identified as containing only 4HB monomers. Similarly,E. coli MBX777 with a plasmid containing hbcT such as pFS16, was grownin LB/4HB (5 μL) and the resuting polymer was identified as PHB4HB with35.5% 4HB monomers.

Example 15 Production of poly(4-hydroxybutyrate) from 4-hydroxybutyratein Recombinant E. coli with No Extrachromosomal DNA

[0110] Poly(4-hydroxybutyrate) can be synthesized from 4-hydroxybutyrateby E. coli expressing 4-hydroxybutyryl-CoA transferase (hbcT) and PHAsynthase (phaC) genes from a plasmid. If these genes are integrated intothe E. coli chromosome and expressed at high levels, the recombinant E.coli should be able to synthesize poly(4-hydroxybutyrate) from4-hydroxybutyrate. The hbcT and phbC genes were inserted into pUTHg(Herrero, et al., J. Bacteriol. 172:6557-67, 1990) as follows. pMSXC₅catand pFS16 were both digested with BamHI and SalI. The large fragment ofpMSXC₅cat and the fragment of pFS16 containing the hbcT gene thusobtained were ligated together using T4 DNA ligase to formpMSXC₅hbcT-cat. The fragment containing the phaC, hbcT, and cat geneswas removed from pMSXC₅hbcT-cat by digestion with AvrII, and it wasinserted using T4 DNA ligase into pUTHg that had been digested withAvrII and treated with calf intestinal alkaline phosphatase to preventself-ligation. The plasmid thus obtained was denoted pMUXC₅hbcT-cat. Theplasmid pMUXC₅hbcT-cat was replicated in MBX129 and conjugated intoMBX1177. The strain MBX1177 is a spontaneous mutant of E. coli strainDH5α that was selected for its ability to grow on minimal4-hydroxybutyrate agar plates. MBX1177 is also naturally resistant tonalidixic acid. The recipient cells were separated from the donor cellsby plating on LB-agar supplemented with 25 μg/mL chloramphenicol and 30μg/mL nalidixic acid. Survivors from this plate were restreaked onminimal medium, containing, per liter: 15 g agar; 2.5 g/L LB powder(Difco; Detroit, Mich.); 5 g glucose; 10 g 4-hydroxybutyrate; 1 mmolMgSO₄; 10 mg thiamine; 0.23 g proline; 25.5 mmol Na₂HPO₄; 33.3 mmolK₂HPO₄; 27.2 mmol KH₂PO₄; 2.78 mg FeSO₄.7H₂O; 1.98 mg MnCl₂.4H₂O; 2.81mg CoSO₄.7H₂O; 0.17 mg CuCl₂.2H₂O; 1.67 mg CaCl₂.2H₂O; 0.29 mgZnSO₄.7H₂O; and 0.5 mg chloramphenicol. Colonies from this plate thatappeared to be especially white and opaque were evaluated in shakeflasks containing the same medium as above except without agar. Theindividual colonies were first grown in 3 mL of LB medium for 8 hours,and 0.5 mL of each culture was used to inoculate 50 mL of the mediumdescribed above. These flasks were incubated at 30° C. for 96 hours. Oneisolate was found by GC analysis (for which the cells were removed fromthe medium by centrifugation for 10 minutes at 2000×g, washed once withwater and centrifuged again, then lyophilized) to contain 4.9%poly(4-hydroxybutate) by weight. This strain was denoted MBX1462 andselected for further manipulations. MBX1462 was treated with the mutagen1-methyl-3-nitro-1-nitrosoguanidine (MNNG), a chemical mutagen, byexposing a liquid culture of MBX1462 to 0.1 mg/mL MNNG for 90 minutes.It was found that 99.8% of the cells were killed by this treatment. Theplating and shake flask experiment described above was repeated, and oneisolate was found by GC analysis to contain 11% poly(4-hydroxybutate) byweight. This strain was denoted MBX1476 and selected for furthermanipulations. The NTG treatment was repeated and killed 96.3% of thecells. The plating and shake flask experiment described above wasrepeated once again, and one isolate was found by GC analysis to contain19% poly(4-hydroxybutate) by weight. This strain was denoted MBX1509.

Example 15 PHBH Copolymer Producing Transgenic E. coli Strains

[0111]E. coli MBX240 is an XL1-blue (Stratagene, San Diego, Calif.)derivative with a chromosomally integrated copy of the PHB polymeraseencoding phbC gene from Ralsionia eutropha. This strain does not formPHAs from carbon sources such as glucose or fatty acids, because of theabsence of enzymes converting acetyl-CoA (generated from carbohydratessuch as glucose) or fatty acid oxidation intermediates, into(R)-3-hydroxyacyl-CoA monomers for polymerization. pMSXJ12 wasconstructed by inserting the phaJ gene from A. caviae digested withEcoRI and PstI into the corresponding sites of pUC18Sfi. The phaJ genewas obtained by polymerase chain reaction using the primers Ac3-5′:5′ AGAATTCAGGAGGACGCCGCATGAGCGCACAATCCCTGG and Ac3-3′:5′ TTCCTGCAGCTCAAGGCAGCTTGACCACG

[0112] using a PCR program including 30 cycles of 45 s at 95 C, 45 s at55 C and 2.5 minutes at 72 C. Transformants of E. coli MBX240 withplasmid pMTXJ12 containing the (R)-specific enoyl-CoA hydratase encodedby the phaj gene from Aeromonas caviae were grown on Luria-Bertanimedium with 10 mM octanoate and 1 mM oleate. After 48 hours of growth,cells were harvested from a 50 ml culture by centrifugation and the cellpellet lyophilized. Lyophilized cells were extracted with chloroform (8ml) for 16 hours and PHA was specifically precipitated from thechloroform solution by adding the chloroform layer to a 10-fold excessethanol. Precipitation was allowed to occur at 4 C and the solid polymerwas air dried and analyzed for composition by acidic butanolysis.Butylated PHA monomers were separated by gas chromatography andidentified the PHA as a poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)copolymer with 2.6% 3-hydroxyhexanoate monomers.

Example 16 Construction of Transgenic E. coli Strains for Screening ofNew and/or Improved Genes for PHA Production

[0113] The phbC gene was introduced into an E. coli cloning strain bybacteriophage P1 transduction. In a procedure similar to that followedfor phbC₅ integration, the phbC gene from R. eutropha was integratedinto the chromosome of MBX23, resulting in MBX143. After chloramphenicolamplification, MBX150, which is resistant to 500 μg/ml chloramphenicol,was isolated. A bacteriophage P1 lysate grown on MBX150 was used totransduce the phbC-cat allele into XL1-Blue [pT7-RecA]. Plasmid pT7RecAexpresses a functional RecA protein which is required for successful P1transduction. The resulting strain MBX240 is an XL1-Blue derivative witha functional PHB polymerase expressed from the chromosome. MBX613 andMBX683 were developed using the same procedures. These strains werederived from MBX245 and XL1-Blue, respectively, and contain integratedAB₅cat (MBX613) or p₁₃AB₅kan (MBX683) operons.

Example 17 Identification of Genes Encoding New, Improved or AncillaryPHA Biosynthetic Enzymes

[0114] MBX240, 613, and 683 are three strains that can be used inscreening procedures for new or improved PHA genes. Using these strains,the following genes have been identified: phbCABFA2 from P. acidophilaand phbCAB from A. latus. In addition, the phaJ gene from A. caviae wasfunctionally expressed in MBX240 to produce PHA from fatty acids.Besides PHA biosynthetic genes specific for C₃ to C₆ monomers, PHAbiosynthetic enzymes for PHAs consisting of medium side-chain 3-hydroxyacids can also be expressed in E. coli. Such strains are useful inidentifying additional PHA biosynthetic enzymes.

Example 18 Integration of pha Genes in R. eutropha

[0115] The plasmids described in the previous examples were used tointegrate pha genes in R. eutropha. Using a PHA-negative mutant of R.eutropha such as #2 (Peoples & Sinskey, J. Biol. Chem. 264:15298-303(1989)) or PHB-4 (Schubert, et al., J. Bacteriol. 170:5837-47 (1988)),PHA formation was restored by integration of phaC from A. caviae incombination with phbAB from Z. ramigera or phaJ from A. caviae. Theresulting strains produced PHAs to variable levels, with a molecularweight in the range of 400,000 to 10,000,000 Da and with a compositionthat includes monomers such as 3-hydroxyhexanoate and3-hydroxyoctanoate.

Example 19 Integration of pha Genes in P. putida

[0116] The plasmids described in the previous examples were used tointegrate pha genes into Pseudomonas putida. The PHA-negative phenotypeof P. putida GPp104 (Huisman et al., J. Biol. Chem. 266:2191-98 (1991))was restored by integration of a phaC3kan cassette where phaC3 encodesthe PHA polymerase from P. oleovorans. Integration of phaC3kan usingpMUXC3kan was also applied to generate mutants of P. putida withmutations in genes encoding enzymes that affect PHA metabolism otherthan phaC. The PHA polymerase gene from A. caviae was also introduced into the chromosome to result in a strain that produces PHAs including3-hydroxy fatty acids in the C₃ to C₉ range.

Example 20 Chromosomal Integration of phaC Genes to Control MolecularWeight of the Resulting PHA

[0117] It is well known that the concentration of PHA polymerasedetermines the molecular weight of the produced PHA when substrate isavailable in excess. Variation of the molecular weight is desirable aspolymer properties are dependent on molecular weight. Chromosomalintegration of phb genes results in variable levels of expression of thepha gene as determined by the chromosomal integration site. It istherefore possible to obtain different transgenic bacteria that havevariable levels of phaC expression and hence produce PHAs of variablemolecular weight. With this system, it is possible to produce PHAs withmolecular weights of greater than 400,000 Da and frequently even inexcess of 1,000,000 Da. This procedure is applicable to anygram-negative bacterium in which the pUT or pLOF derived plasmids can beintroduced, such as E. coli, R. eutropha, P. putida, Klebsiellapneumoniae, Alcaligenes latus, Azotobacter vinelandii, Burkholderiacepacia, Paracoccus denitrificans and in general in species of theEscherichia, Pseudomonas, Ralstonia, Burkholderia, Alcaligenes,Klebsiella, Azotobacter genera.

Example 21 Integration of the PHB Genes as a Single Operon

[0118] A plasmid, pMSXABC₅kan, was constructed such that the thiolase(phbA), reductase (phbB), and PHB synthase (phbC) genes from Zoogloearamigera and the kanamycin resistance gene (kan) were linked as anoperon in the vector pUC18Sfi. This expression cassette was then excisedas an AvrII fragment and inserted into the AvrII site of pUT to obtainpMUXABC₅kan.

[0119] S17-1 λpir strains with pMUXABC₅kan were mated with MBX247.Transgenic strains in which phbABC₅kan had integrated into thechromosome were selected on LB/N1/Km plates. Among the integrants, PHBproducers were identified on LB/glucose plates. One strain thusconstructed, MBX1164, was selected for further study.

[0120] Thiolase (Nishimura et al., 1978, Arch. Microbiol. 116:21-24) andreductase (Saito et al., 1977, Arch. Microbiol. 114:211-217) assays wereconducted on MBX1164 crude extracts. The cultures were grown in 50 mL of0.5×E2 medium supplemented with 20 g/L glucose. One unit (U) was definedas the amount of enzyme that converted 1 82 mol of substrate to productper min. 3-Ketothiolase activity was determined to be 2.23±0.38 and2.48±0.50 U/mg in two independent trials, and 3-hydroxybutyryl-CoAreductase activity was determined to be 4.10±1.51 and 3.87±0.15 U/mg intwo independent trials.

[0121] Strain MBX1164 was evaluated for its PHB-producing ability insquare shake bottles. The cells were grown in 2 mL of LB, and 0.1 mL ofthis was used as an inoculum for the 50-mL shake bottle culture. Theshake bottle contained E2 medium supplemented with 0.25% corn steepliquor (Sigma, St. Louis, Mo.) and 20 g/L glucose. After incubation at30° C. for 48 hours with shaking at 200 rpm, the biomass concentrationhad reached 2.6 g/L, and the PHB concentration had reached 11.7 g/L;thus the cells contained 82% PHB by weight.

Example 22 Integration of the Pseudomonas oleovorans PHA Synthase intothe E. coli Chromosome

[0122] A PHA synthase (phaC) cassette from the P. oleovorans chromosomeand a promoterless chloramphenicol resistance gene were inserted intopUC118 such that an operon of the two genes was formed; i.e., they wereoriented in the same direction and could be transcribed on the samemRNA. The sequence of the P. oleovorans phaC gene is shown below. ThephaC-cat operon was excised from this plasmid by digestion with KpnI andHindIII and ligated to pUC18SfiI that had been digested with the sametwo enzymes to form pMSXC₃cat. This allowed the phaC-cat operon to beflanked by AvrII sites. The phaC-cat operon was removed from pMSXC₃catby digestion with AvrII and FspI. Because the two AvrII fragments ofpMSXC₃cat were nearly the same size, FspI was used to facilitateisolation of the phaC-cat operon by cutting the rest of the vector intotwo pieces. The AvrII fragment was ligated to pUTkan which had beendigested with AvrII and treated with alkaline phosphatase to preventself-ligation. The plasmid thus produced was denoted pMUXC₃cat. Theoperon on this plasmid actually consisted of phaC-cat-kan. Strain CC118λpir (a λpir lysogenic strain) was transformed with pMUXC₃cat to producestrain MBX130. Equal amounts of strains MBX130 and MBX245 were mixed onan LB agar plate and incubated for 8 hours at 37° C. The mixed cellswere then used as an inoculum for an overnight 37° C. culture ofLB-chloramphenicol (25 μg/mL)-nalidixic acid (30 μg/mL). Single colonieswere isolated from this culture by plating on LB-chloramphenicol (25μg/mL)-nalidixic acid (30 μg/mL)-kanamycin (25 μg/mL). The colonies thusisolated have a transducible phaC-cat-kan cassette on the chromosome, asshown by the ability to use P1 transduction to introduce the cassetteinto the chromosome of other strains and select for resistance to bothchloramphenicol and kanamycin. Pseudomonas oleovorans PHA synthase(phaC).ATGAGTAACAAGAACAACGATGAGCTGCAGCGGCAGGCCTCGGAAAACACCCTGGGGCTGAACCCGGTCATCGGTATCCGCCGCAAAGACCTGTTGAGCTCGGCACGCACCGTGCTGCGCCAGGCCGTGCGCCAACCGCTGCACAGCGCCAAGCATGTGGCCCACTTTGGCCTGGAGCTGAAGAACGTGCTGCTGGGCAAGTCCAGCCTTGCCCCGGAAAGCGACGACCGTCGCTTCAATGACCCGGCATGGAGCAACAACCCACTTTACCGCCGCTACCTGCAAACCTATCTGGCCTGGCGCAAGGAGCTGCAGGACTGGATCGGCAACAGCGACCTGTCGCCCCAGGACATCAGCCGCGGCCAGTTCGTCATCAACCTGATGACCGAAGCCATGGCTCCGACCAACACCCTGTCCAACCCGGCAGCAGTCAAACGCTTCTTCGAAACCGGCGGCAAGAGCCTGCTCGATGGCCTGTCCAACCTGGCCAAGGACCTGGTCAACAACGGTGGCATGCCCAGCCAGGTGAACATGGACGCCTTCGAGGTGGGCAAGAACCTGGGCACCAGTGAAGGCGCCGTGGTGTACCGCAACGATGTGCTGGAGCTGATCCAGTACAACCCCATCACCGAGCAGGTGCATGCCCGCCCGCTGCTGGTGGTGCCGCCGCAGATCAACAAGTTCTACGTATTCGACCTGAGCCCGGAAAAGAGCCTGGCACGCTACTGCCTGCGCTCGCAGCAGCAGACCTTCATCATCAGCTGGCGCAACCCGACCAAAGCCCAGCGCGAATGGGGCCTGTCCACCTACATCGACGCGCTCAAGGAGGCGGTCGACGCGGTGCTGGCGATTACCGGCAGCAAGGACCTGAACATGCTCGGTGCCTGCTCCGGCGGCATCACCTGCACGGCATTGGTCGGCCACTATGCCGCCCTCGGCGAAAACAAGGTCAATGCCCTGACCCTGCTGGTCAGCGTGCTGGACACCACCATGGACAACCAGGTCGCCCTGTTCGTCGACGAGCAGACTTTGGAGGCCGCCAAGCGCCACTCCTACCAGGCCGGTGTGCTCGAAGGCAGCGAGATGGCCAAGGTGTTCGCCTGGATGCGCCCCAACGACCTGATCTGGAACTACTGGGTCAACAACTACCTGCTCGGCAACGAGCCGCCGGTGTTCGACATCCTGTTCTGGAACAACGACACCACGCGCCTGCCGGCCGCCTTCCACGGCGACCTGATCGAAATGTTCAAGAGCAACCCGCTGACCCGCCCGGACGCCCTGGAGGTTTGCGGCACTCCGATCGACCTGAAACAGGTCAAATGCGACATCTACAGCCTTGCCGGCACCAACGACCACATCACCCCGTGGCAGTCATGCTACCGCTCGGCGCACCTGTTCGGCGGCAAGATCGAGTTCGTGCTGTCCAACAGCGGCCACATCCAGAGCATCCTCAACCCGCCAGGCAACCCCAAGGCGCGCTTCATGACCGGTGCCGATCGCCCGGGTGACCCGGTGGCCTGGCAGGAAAACGCCACCAAGCATGCCGACTCCTGGTGGCTGCACTGGCAAAGCTGGCTGGGCGAGCGTGCCGGCGAGCTGAAAAAGGCGCCGACCCGCCTGGGCAACCGTGCCTATGCCGCTGGCGAGGCATCCCCGGGCACCTACGTTCACGAGCGTTGA

[0123] Modifications and variations of the present invention will beobvious to those of skill in the art from the foregoing detaileddescription. Such modifications and variations are intended to comewithin the scope of the following claims.

1 39 1 50 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 1 aagcttccca gcccgctaat gagcgggctt ttttttgaac aaaagcatgc50 2 71 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 2 gtctacgtac cccaggcttt acatttatgc ttccggctcg tatgttgtgtggaattgtga 60 gcggttcgga c 71 3 48 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide 3 agcttcccag cccgctaatgagcgggcttt tttttgaaca aaagctgc 48 4 40 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 4 cttttgttcaaaaaaaagcc cgctcattag cgggctggga 40 5 45 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 5 ggctcgtataatgtgtggag ggagaaccgc cgggctcgcg ccgtt 45 6 53 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 6 ctagaacggcgcgagcccgg cggttctccc tccacacatt atacgagcct gca 53 7 43 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 7 ttcagaaaattattttaaat ttcctcttga catttatgct gca 43 8 43 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 8 gcataaatgtcaagaggaaa tttaaaataa ttttctgaat gca 43 9 58 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 9 ctagtgccggacccggttcc aaggccggcc gcaaggctgc cagaactgag gaagcaca 58 10 56 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide10 tatgtgcttc ctcagttctg gcagccttgc ggccggcctt ggaaccgggt ccggca 56 1126 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 11 tcccctgtca taaagttgtc actgca 26 12 26 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 12gtgacaactt tatgacaggg gatgca 26 13 19 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 13 tgaccaacatacgagcggc 19 14 20 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide 14 ctaccagaac tttgctttcc 20 15 36 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide15 tgcatgcgat atcaattgtc cagccagaaa gtgagg 36 16 20 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 16atttattcaa caaagccgcc 20 17 38 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 17 cccagcccgc taatgagcgg gcttttttttgaacaaaa 38 18 44 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide 18 tacgtatttt gttcaaaaaa aagcccgctc attagcgggctggg 44 19 63 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 19 tacgtacccc aggctttaca tttatgcttc cggctcgtatgttgtgtgga attgtgagcg 60 gtt 63 20 61 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 20 ttcgaaccgctcacaattcc acacaacata cgagccggaa gcataaatgt aaagcctggg 60 g 61 21 14 DNAPseudomonas oleovorans 21 aggaggtttt tatg 14 22 42 DNA ArtificialSequence Description of Artificial Sequence primer 22 ggaattcaggaggttttatg gcgaccggca aaggcgcggc ag 42 23 38 DNA Artificial SequenceDescription of Artificial Sequence primer 23 gctctagaag ctttcatgccttggctttga cgtatcgc 38 24 42 DNA Artificial Sequence Description ofArtificial Sequence primer 24 ggaattcagg aggttttatg agtaacaagaacaacgatga gc 42 25 38 DNA Artificial Sequence Description of ArtificialSequence primer 25 gctctagaag ctttcaacgc tcgtgaacgt aggtgccc 38 26 42DNA Artificial Sequence Description of Artificial Sequence primer 26ggaattcagg aggttttatg agccaaccat cttatggccc gc 42 27 38 DNA ArtificialSequence Description of Artificial Sequence primer 27 gctctagaagctttcatgcg gcgtcctcct ctgttggg 38 28 42 DNA Artificial SequenceDescription of Artificial Sequence primer 28 ggaattcagg aggttttatgaggccagaaa tcgctgtact tg 42 29 38 DNA Artificial Sequence Description ofArtificial Sequence primer 29 gctctagaag ctttcagatg gcaaatgcat gctgcccc38 30 43 DNA Artificial Sequence Description of Artificial Sequenceprimer 30 aggagctcag gaggttttat gagcgcacaa tccctggaag tag 43 31 38 DNAArtificial Sequence Description of Artificial Sequence primer 31gctctagaag cttttaaggc agcttgacca cggcttcc 38 32 43 DNA ArtificialSequence Description of Artificial Sequence primer 32 ggaattcaggaggttttatg acgcgtgaag tggtagtggt aag 43 33 37 DNA Artificial SequenceDescription of Artificial Sequence primer 33 gctctagaag ctttcagatacgctcgaaga tggcggc 37 34 42 DNA Artificial Sequence Description ofArtificial Sequence primer 34 ggaattcagg aggttttatg atcctcaccccggaacaagt tg 42 35 38 DNA Artificial Sequence Description of ArtificialSequence primer 35 gctctagaag ctttcagggc actaccttca tcgttggc 38 36 38DNA Artificial Sequence Description of Artificial Sequence primer 36gctctagaag ctttcaggca gccgtcgtct tctttgcc 38 37 39 DNA Aeromonas caviae37 agaattcagg aggacgccgc atgagcgcac aatccctgg 39 38 29 DNA Aeromonascaviae 38 ttcctgcagc tcaaggcagc ttgaccacg 29 39 1680 DNA Pseudomonasoleovorans 39 atgagtaaca agaacaacga tgagctgcag cggcaggcct cggaaaacaccctggggctg 60 aacccggtca tcggtatccg ccgcaaagac ctgttgagct cggcacgcaccgtgctgcgc 120 caggccgtgc gccaaccgct gcacagcgcc aagcatgtgg cccactttggcctggagctg 180 aagaacgtgc tgctgggcaa gtccagcctt gccccggaaa gcgacgaccgtcgcttcaat 240 gacccggcat ggagcaacaa cccactttac cgccgctacc tgcaaacctatctggcctgg 300 cgcaaggagc tgcaggactg gatcggcaac agcgacctgt cgccccaggacatcagccgc 360 ggccagttcg tcatcaacct gatgaccgaa gccatggctc cgaccaacaccctgtccaac 420 ccggcagcag tcaaacgctt cttcgaaacc ggcggcaaga gcctgctcgatggcctgtcc 480 aacctggcca aggacctggt caacaacggt ggcatgccca gccaggtgaacatggacgcc 540 ttcgaggtgg gcaagaacct gggcaccagt gaaggcgccg tggtgtaccgcaacgatgtg 600 ctggagctga tccagtacaa ccccatcacc gagcaggtgc atgcccgcccgctgctggtg 660 gtgccgccgc agatcaacaa gttctacgta ttcgacctga gcccggaaaagagcctggca 720 cgctactgcc tgcgctcgca gcagcagacc ttcatcatca gctggcgcaacccgaccaaa 780 gcccagcgcg aatggggcct gtccacctac atcgacgcgc tcaaggaggcggtcgacgcg 840 gtgctggcga ttaccggcag caaggacctg aacatgctcg gtgcctgctccggcggcatc 900 acctgcacgg cattggtcgg ccactatgcc gccctcggcg aaaacaaggtcaatgccctg 960 accctgctgg tcagcgtgct ggacaccacc atggacaacc aggtcgccctgttcgtcgac 1020 gagcagactt tggaggccgc caagcgccac tcctaccagg ccggtgtgctcgaaggcagc 1080 gagatggcca aggtgttcgc ctggatgcgc cccaacgacc tgatctggaactactgggtc 1140 aacaactacc tgctcggcaa cgagccgccg gtgttcgaca tcctgttctggaacaacgac 1200 accacgcgcc tgccggccgc cttccacggc gacctgatcg aaatgttcaagagcaacccg 1260 ctgacccgcc cggacgccct ggaggtttgc ggcactccga tcgacctgaaacaggtcaaa 1320 tgcgacatct acagccttgc cggcaccaac gaccacatca ccccgtggcagtcatgctac 1380 cgctcggcgc acctgttcgg cggcaagatc gagttcgtgc tgtccaacagcggccacatc 1440 cagagcatcc tcaacccgcc aggcaacccc aaggcgcgct tcatgaccggtgccgatcgc 1500 ccgggtgacc cggtggcctg gcaggaaaac gccaccaagc atgccgactcctggtggctg 1560 cactggcaaa gctggctggg cgagcgtgcc ggcgagctga aaaaggcgccgacccgcctg 1620 ggcaaccgtg cctatgccgc tggcgaggca tccccgggca cctacgttcacgagcgttga 1680

We claim:
 1. A genetically engineered microorganism having at least onegene involved in synthesis of polyhydroxyalkanoates selected from thegroup consisting of thiolase, reductase, PHB synthase, PHA synthase,acyl-CoA transferase, enoyl-CoA hydratase, integrated into thechromosome, which produce polyhydroxyalkanoate.
 2. The microorganism ofclaim 1 selected from the group consisting of E. coli, Alcaligeneslatus, Alcaligenese eutrophus, Azotobacter, Pseudomonas putida, andRalstonia eutropha.
 3. The microorganism of claim 1 wherein the gene isinserted using transposan mutagenesis.
 4. The microorganism of claim 1comprising multiple genes involved in synthesis of polyhydroxyalkanoatewherein the genes are integrated operably linked as an operon.
 5. Themicroorganism of claim 1 wherein the gene is integrated operably linkedunder the control of a promoter.
 6. The microorganism of claim 1 whereinthe gene is integrated operably linked with upstream activatingsequences
 7. The microorganism of claim 1 wherein the gene is integratedoperably linked with mRNA stabilizing sequences.
 8. The microorganism ofclaim 5 wherein the gene is operably linked with promoter including aconsensus E. coli pho box and −35 promoter region that is regulated bythe phosphate concentration in the medium.
 9. The microorganism of claim5 wherein the promoter is selected from the group consisting of promoterthat induces expression under general stress conditions such as nutrientlimitation, pH or heat shock, and administration of toxic chemicals. 10.The microorganism of claim 1 wherein the gene is integrated operablylinked with a selection marker.
 11. The microorganism of claim 1 whereinthe gene is isolated or derived from a microorganism selected from thegroup consisting of A. eutrophus, Aeromonas caviae, Zoogloea ramigera,Nocardia, Rhodococcus, Pseudomonas Sp. 61-3, Pseudomonas acidophila,Pseudomonas oleovarans, Chromobacterium violaceum, and Alcaligeneslatus.
 12. The microorganism of claim 1 wherein the gene is selectedfrom the group consisting of PHB polymerase from R. eutropha (C₁), PHApolymerase from P. oleovorans (C3), PHB polymerase from A. caviae (C12),ACP::CoA transacylase from P. putida (G3), (R)-specific enouyl-CoAhydratase from A. caviae (J12), a broad substrate specific3-ketoacyl-CoA thiolase from R. eutropha (A1-II), and phasins from R.eutropha (P1-I and P1-II).
 13. The microorganism of claim 1 wherein thegene is integrated as a single copy on the chromosome of themicroorganism.
 14. A method for screening for a gene involved insynthesis of polyhydroxyalkanoates that enhances production comprisingmutating a genetically engineered microorganism having at least one geneinvolved in synthesis of polyhydroxyalkanoates selected from the groupconsisting of thiolase, reductase, PHB synthase, PHA synthase, acyl-CoAtransferase, enoyl-CoA hydratase, integrated into the chromosome, whichproduces polyhydroxyalkanoate, and screening for enhanced production ofpolyhydroxyalkanoates.
 15. The method of claim 14 wherein the gene isisolated or derived from a microorganism selected from the groupconsisting of A. eutrophus, Aeromonas caviae, Zoogloea ramigera,Nocardia, Rhodococcus, Pseudomonas Sp. 61-3, Pseudomonas acidophila,Pseudomonas oleovarans, Chromobacterium violaceum, and Alcaligeneslatus.
 16. The method of claim 14 wherein the microorganisms are missingone or more genes required for production of polyhydroxyalkanoates. 17.The method of claim 14 wherein the microorganims producepolyhydroxyalkanoates, further comprising selecting genes which resultin increased polyhydroxyalkanoate production.
 18. A method for producingpolyhydroxyalkanoates comprising culturing genetically engineeredmicroorganisms having at least one gene involved in synthesis ofpolyhydroxyalkanoates selected from the group consisting of thiolase,reductase, PHB synthase, PHA synthase, acyl-CoA transferase, enoyl-CoAhydratase, integrated into the chromosome, with appropriate substrateunder conditions wherein the microorganisms producepolyhydroxyalkanoate.
 19. The method of claim 18 wherein themicroorganisms comprise multiple genes involved in synthesis ofpolyhydroxyalkanoate wherein the genes are integrated operably linked asan operon.
 20. The method of claim 18 wherein the microorganismscomprise genes selected from the group consisting of genes integratedoperably linked under the control of a promoter, genes integratedoperably linked with upstream activating sequences, genes integratedoperably linked with mRNA stabilizing sequences, genes operably linkedwith a promoter including a consensus E. coli pho box and −35 promoterregion that is regulated by the phosphate concentration in the medium,and genes integrated operably linked with a selection marker.